Effect of corpus cardiacum extracts on the ATPase activity of locust rectum

The Mg²⁺ dependent and Na⁺K⁺-activated ATPase activities of microsomal preparations from the rectum of Locusta migratoria were both stimulated, to varying extents, by crude extracts of the corpora cardiaca of this species. Mg²⁺ ATPase activity increased by approximately 549% whereas the hormonal stimulation of Na⁺K⁺-activated ATPase depended upon the concentration of sodium and potassium ions. At 100 mM Na⁺ and 20 mM K⁺, conditions which approximate to optimum for this enzyme system, Na⁺K⁺-activated ATPase activity increased by about 14%. At sub-optimum concentrations of these ions, i.e. 50 and 5 mM Na⁺ and K⁺ respectively, the increase in Na⁺K⁺-activated ATPase activity was about 205%. Ouabain at a concentration of 10^?3 M completely abolished this stimulated activity and was consistently effective in partially reducing the stimulation of Mg²⁺ ATPase activity by corpora cardiaca extracts.     

Sensitivity of ATPase activities to fire ant venom and abdomen preparations.

The sensitivity of fire ant, $\text{Solenopsis richteri}$ (Forel), head homogenate ATPase to its venom and to a cyclohexane extract of whole fire ants were investigated. Na⁺K⁺ and oligomycin-sensitive Mg²⁺ ATPase activities were inhibited by both preparations. Oligomycin-insensitive Mg²⁺ ATPase activity was inhibited by low concentrations but showed strong stimulation at high concentrations of the venom preparations. Lineweaver-Burk plots of enzyme data in the presence or absence of inhibitor indicated that the inhibitor action was non-competitive with ATP for Na⁺K⁺ and oligomycin-sensitive Mg²⁺ ATPase activities. However, the oligomycin-insensitive Mg²⁺ ATPase activity showed a mixed type response to the inhibitor. Tests on pure samples of known venom components indicate that they cause the observed effects on the ATPase activities.     

ATPases in rabbit erythrocytes: stimulation by HCO3-anion and by Na-cation-plus-K-cation.

A Mg²⁺Na⁺K⁺ATPase was found in a ghost preparation from rabbit erythrocytes, a finding in conflict with previous reports, but in agreement with the known kinetics of cation movements in these cells. However the Mg²⁺Na⁺K⁺ATPase was not inhibited by 10⁻⁴M ouabain, nor by 10⁻⁴M Ca²⁺. The physiological status of this enzyme is discussed. The basic Mg²⁺-ATPase activity in this preparation is also stimulated by HCO₃⁻; it is suggested that the HCO₃⁻-stimulated ATPases reported in a variety of other preparations are not necessarily due to mitochondrial contamination but could well originate from the plasma membrane.     

Stimulation of (Na+K+)-ATPase of rat liver plasma membrane by amino acids

The effect of twelve l-amino acids on the activity of liver plasma membrane (Na⁺K⁺)-ATPase has been tested. Histidine and arginine significantly enhanced the activity. The activtion by histidine showed saturation kinetics with an apparent K_a of about 8 mM, and was evident over a wide range of Na⁺ concentrations. The same amino acid did not significantly affect the Mg²⁺-dependent ATPase activity.     

Relationship of Na+-K+-activated ATPase to fluid production by Malpighian tubules of Locusta migratoria.

The presence of a Na⁺K⁺-activated, Mg²⁺-dependent ATPase (E.C. 3.6.1.3) has been demonstrated in microsomal preparations from the Malpighian tubules of Locusta. The effects of sodium and potassium ions, and different concentrations of ouabain, have been studied in relation to the activity of this enzyme and the ability of in vitro Malpighian tubule preparations to secrete fluid. From these studies it seems highly likely that a Na⁺K⁺ activated ATPase ‘pump’ is involved in fluid transport across the walls of the tubules.     

Properties and nature of (Na+ + Mg2+)-dependent adenosine triphosphatase in the gills of the eel, angvilla anguilla (L.)

The specific activity of (Na⁺ + Mg²⁺)-dependent ATPase is three times greater in the microsomes of sea-water eels than in freshwater eels; the specific activity is one quarter of that of (Na⁺ + K⁺ + Mg²⁺)-dependent ATPase in both cases.(Na⁺ + Mg²⁺)-dependent ATPase is optimally active in a medium containing 8 mM NaCl, 4 mM MgCI₂, 4 mM ATP, pH 8.8 and at 30 °C; the enzyme is inhibited by ouabain, by NaCl concentrations > 100 mM and by treatment with urea.It is concluded that the (Na⁺ + Mg²⁺)-dependent ATPase activity of gills arises from the presence of a (Na⁺ + K⁺ + Mg²⁺)-dependent ATPase.     

Localization of various ATPases in fresh and cryopreserved bovine spermatozoa

Different ATPases may control the various functional changes that spermatozoa undergo just prior to fertilization, with the enzyme's specific location within the cell reflecting its function. The activities of Mg²⁺-ATPase, Ca²⁺Mg²⁺-ATPase, Na⁺K⁺-ATPase and Ca²⁺-ATPase were determined for head plasma membranes (HPM) and sperm body membrane (SBM) from both fresh (n = 4) and cryopreserved bovine spermatozoa (n = 4) and fresh homogenized whole spermatozoa (HWS) (n = 6). No activity of Ca²⁺Mg²⁺-ATPase was found in any preparation from spermatozoa. Ca²⁺-ATPase was detected in fresh SBM and HWS but not in HPM. Activity of Mg²⁺-ATPase and Na⁺K⁺-ATPase was higher in HPM than HWS or SBM (P < 0.01). Cryopeserving the whole sperm reduced the activities of all three enzymes, but Na⁺K⁺-ATPase was more sensitive to cryopreservation than Mg²⁺-ATPase (P ≤ 0.05). Enzyme location suggests that Ca²⁺-ATPase may be associated with events in the flagellum, while Mg²⁺-ATPase and Na⁺K⁺-ATPase may affect functions in the sperm head. Cryopreservation-induced damage to ATPases might be involved in reducing the fertilizing ability of cryopreserved spermatozoa.     

Characterization of microsomal ATPases from developing human placenta

Activities and some properties of microsomal ATPases have been studied in developing human placenta. The enzyme activities (Na⁺ + K⁺ + Mg²⁺, Mg²⁺, and Ca²⁺ dependent) in the placenta increase steadily with gestational age until the 18th to 21st week, and decrease in the second half of pregnancy. Mg²⁺-dependent and Na⁺ + K⁺ + Mg²⁺-dependent ATPases possess nearly the same K_m (apparent) for ATP, while the Ca²⁺-dependent enzyme shows a different one. Mg²⁺-dependent ATPase shows higher substrate affinity than Ca²⁺-dependent ATPase, although the V_max of the Mg²⁺-dependent enzyme is lower than that of the latter. However, for each enzyme, the K_m remains almost constant and V_max varies during ontogenic development. V_max of the enzymes decline at term. The enzymes are heat-labile, unaffected by amino acids, namely, l-phenylalanine, l-leucine, and l-tryptophan, and deoxycholate inhibits the enzyme activities by about 50%.     

Elevation of Different Ion-Specific ATPase Activities by L-Thyroxine (T4) in Different Tissues of Tasar Silkworm, Antheraea mylitta (Lepidoptera: Saturniidae) during Developmental Stages

Tissue-specific age-dependent changes were observed in Na⁺K⁺-, Ca²⁺-, and Mg²⁺-ATPase activities in tropical tasar silkworm, Antheraea mylitta Drury. Maximum enzyme activity was recorded in all the tissues on day 12 (before spinning) in control group of animals. In testis, Na⁺K⁺-, Ca²⁺-, and Mg²⁺-ATPase activities gradually increased from day 2 to day 12 during fifth larval age and level was maintained up to adult eclosion while, in ovary, a marked decline was noted up to day of adult emergence. Further, a significant and sharp rise was found in ATPase activity in silk gland tissue up to day 12 and afterwards a drastic fall was noted on day 15 (end of spinning) during fifth larval age.Administration of T4 to fifth stage larvae (1 hr old) at doses 0.5–2.0 μg/g significantly elevated the Na⁺K⁺-, Ca²⁺-, and Mg²⁺-ATPase activities in larval and pupal gonads in a dose-dependent fashion. But, in moths, the enhancement was very much confined to Na⁺K⁺- and Ca²⁺-ATPase in testes and only Ca²⁺-ATPase in ovaries. Again, in silk glands thyroxine (0.5–2.0 μg/g) caused a significant rise in the all ion-dependent ATPase activities only during the fifth larval stage. Interestingly, higher doses of T4 (4.0 μg/g) caused a significant reduction in Na⁺K⁺-, Ca²⁺- and Mg²⁺-ATPase in all the tissues almost all the days studied so far. However, lower doses of T4 (0.1 and 0.25 μg/g) remained ineffective in altering the different ion-specific ATPase activities. This study suggests, that mammalian thyroxine has a metabolic influence showing biphasic nature of action in tasar silkworm ATPase system.     

Properties of (Na+ + K+)-activated ATPase in rat liver plasma membranes enriched with bile canaliculi

Liver plasma membranes enriched in bile canaliculi were isolated from rat liver by a modification of the technique of Song et al. (J. Cell Biol. (1969) 41, 124–132) in order to study the possible role of ATPase in bile secretion. Optimum conditions for assaying (Na⁺ + K⁺)-activated ATPase in this membrane fraction were defined using male rats averaging 220 g in weight. (Na⁺ + K⁺)-activated ATPase activity was documented by demonstrating specific cation requirements for Na⁺ and K⁺, while the divalent cation, Ca²⁺, and the cardiac glycosides, ouabain and scillaren, were inhibitory. (Na⁺ + K⁺)-activated ATPase activity averaged 10.07 ± 2.80 μmol P_i/mg protei per h compared to 50.03 ± 11.41 for Mg²⁺-activated ATPase and 58.66 ± 10.07 for 5′-nucleotidase. Concentrations of ouabain and scillaren which previously inhibited canalicular bile secretion in the isolated perfused rat liver produced complete inhibition of (Na⁺ + K⁺)-activated ATPase without any effect on Mg²⁺-activated ATPase. Both (Na⁺ + K⁺)-activated ATPase and Mg²⁺-activated ATPase demonstrated temperature dependence but differed in temperature optima. Temperature induced changes in specific activity of (Na⁺ + K⁺)-activated ATPase directly paralleled previously demonstrated temperature optima for bile secretion. These studies indicate that (Na⁺ + K⁺)-activated ATPase is present in fractions of rat liver plasma membranes that are highly enriched in bile canaliculi and provide a model for further study of the effects of various physiological and chemical modifiers of bile secretion and cholestasis.     

Ca-Mg-ATPase activity in brain coated microvesicles purified on immunosorbents

Coated microvesicle fractions isolated from ox forebrain cortex by the ultracentrifugation procedure of Pearse (1) and by the modified, less time consuming method of Keen et al (2) had comparable Ca²⁺+Mg²⁺ dependent ATPase activities (about 9 μmol/h per mg protein). The Na⁺+K⁺+Mg²⁺ dependent ATPase activity was 3.2 μmol/h per mg (±1.0, S.D., n=3) when microvesicles were prepared according to (1) and 1.5 μmol/h per mg (±1.0, S.D., n=3) when prepared according to (2).Oligomycin, ruthenium red, and trifluoperazine, inhibitors of Ca²⁺ transport in mitochondria and erythrocyte membranes had no effect on Ca²⁺+Mg²⁺ dependent ATPase from any of the preparations.As demonstrated both by ATPase assays and electron microscopy, coated microvesicles could be bound to immunosorbents prepared with poly-specific antibodies against a coated microvesicle fraction obtained by the method of Pearse (1). The binding could be inhibited by dissolved coat protein using partially purified clathrin. The fraction of coated vesicles eluted from the immunosorbent was purified relative to the starting material as judged by electron microscopy.The Ca²⁺+Mg²⁺ ATPase activity and calmodulin content was copurified with the coated microvesicles and the specific activity of Na⁺+K⁺+Mg²⁺ ATPase was decreased.Na⁺+K⁺+Mg²⁺ dependent ATPase activity in the coated microvesicle fraction could be ascribed to membranes with the appearance of microsomes. These membranes were also bound to the immunosorbents, but the binding was not influenced by clathrin. The capacity of the immunosorbents for these membranes was less than for the coated microvesicles, resulting in a decrease of Na⁺+K⁺+Mg²⁺ dependent ATPase activity in the eluted coated microvescile fraction.It was concluded that Ca²⁺+Mg²⁺ ATPase activity is not a contamination from plasma membrane vesicles or mitochondrial membranes but seems to be an integral part of the coated vesicle membrane.     

Direct stimulation of human erythrocyte membrane (Na+ + k+)-mg ATPase activity in vitro by physiological concentrations of d-aldosterone.

The effect of d-aldosterone on human erythrocyte ghost (Na⁺ + K⁺)-Mg ATPase has been studied. Aldosterone at 3.225 × 10^?10M caused a 450% activation of (Na⁺ + K⁺)-Mg ATPase activity whilst inhibiting (Na⁺ + Na⁺)-Mg ATPase activity. Aldosterone acts by reducing the affinity of the external K⁺ site of (Na⁺ + K⁺)Mg ATPase for Na⁺ thereby resulting in improved efficiency of Na⁺ ? K⁺ transfer. Aldosterone was additionally found to modify both the Na⁺ and K⁺ activation of (Na⁺ + K⁺)Mg ATPase incubated in the presence of commercial ATP containing orthovanadate. Aldosterone was found to reverse the inhibitory effects of orthovanadate at high Na⁺ and K⁺ concentrations. The physiological significance of orthovanadate and aldosterone are discussed.     

Effects of delta9-tetrahydrocannabinol on ATPases in mouse brain subcellular fractions.

The effect of (?)-Δ⁹-THC on the activities of Mg²⁺?, Na⁺?K⁺? and Mg²⁺Ca²⁺-ATPases were studied in mouse brain subcellular fractions. $\text{In vitro}$treatment with Δ⁹-THC produced a dose dependent stimulation of Mg²⁺ ATPase in the crude mitochondrial fraction and its subfractions and a dose-related inhibition of this activity in the microsomal fraction. Na⁺-K⁺- and Mg²⁺-Ca²⁺-ATPase activities were inhibited in a dose-related manner in all subcellular fractions studied.     

Effect of the diazonium salt of sulfanilic acid on human erythrocyte ATPase activity

External treatment of human erythrocytes with the diazonium salt of sulfanilic acid does not inhibit the Mg²⁺-dependent ATPase but does markedly inhibit the Ca²⁺-stimulated ATPase activity. Inhibition of the (Na⁺ + K⁺)-dependent activity is dependent upon the concentration of diazonium salt used. Treatment of membrane fragments does not irreversibly inhibit the (Na⁺ + K⁺)-dependent ATPase even though the diazonium salt binds covalently to membrane components. However, the Mg²⁺-dependent and Ca²⁺-stimulated ATPase activities are irreversibly inhibited. ATP and Mg-ATP will completely protect the (Na⁺ + K⁺)-dependent ATPase when present during treatment of membrane fragments with the diazonium salt, but only Mg-ATP will protect the Mg²⁺-dependent ATPase from inhibition. The Ca²⁺-stimulated ATPase activity is not protected.     

Variable ATPase composition of human tumor plasma membranes

Purified plasma membranes from several transplantable human tumors exhibit very high Mg²⁺-dependent ATPase activities. Three types of Mg²⁺-dependent ATPases can be demonstrated: (1) an ouabain sensitive Na⁺, K⁺-ATPase, which is a minor component of the tumor plasma membrane ATPase, (2) a Mg²⁺-activated ATPase, which is a non-specific nucleoside triphosphatase, and (3) an ATPase activity stimulated by Na⁺ (or K⁺) alone. In three human melanomas, only the first two activities are found. In an astrocytoma and an oat cell carcinoma, all three activities are found. In the same two tumors, the plasma membrane Mg²⁺-ATPase is also stimulated by Con A. The relationship of these ATPases are discussed.     

Angiotensin increases microsomal (Na−K-ATPase activity in several tissues

Angiotensin increased microsomal (Na⁺K⁺)-activated ATPase from rat hypothalamus, mucosa of colon and bovine adrenal cortex but not brain cortex. Angiotensin did not change significantly Mg²⁺-ATPase in these tissues. Enhancement by angiotensin was evident at concentrations of 10⁻⁸ − 10⁻¹² M. In bovine adrenal cortex angiotensin increased ATPase activity in the outer and not in the inner layer. Angiotensin increased ATPase activity with Na⁺ above 20 mM and K⁺ above 5 mM present simultaneously, but not with either ion alone.     

The mode of activation by potassium of potassium-dependent and ouabain-sensitive phosphatase activity.

A new simple procedure has been developed for the purification of plasma membranes from rabbit kidney microsomes which yields a three- to fourfold increase in the specific activity of Na⁺-K⁺-adenosine triphosphatase (ATPase). The procedure differs from previous methods with deoxycholate or other detergents and does not change the molecular activity of the ATPase. The K⁺-dependent p-nitrophenylphosphatase activity of the native Na⁺-K⁺-ATPase is controlled more effectively by Mg²⁺ in the presence of K⁺ at concentrations higher than that of Mg²⁺, and by K⁺ in the presence of Mg²⁺ at concentrations higher than that of K⁺. The enzyme in its Mg²⁺-regulating state, which shows K⁺-saturation curves with a Hill coefficient of 1, is less sensitive to ouabain (I_0.5 = 90 μM) and corresponds to the enzyme conformation reported previously which is inhibited by the concurrent presence of Na⁺ and ATP or of Na⁺ and oligomycin (I_0.5 is the midpoint of the saturation curve). The enzyme in its K⁺-regulating state, which shows K⁺-saturation curves with a Hill coefficient of 2, is more sensitive to ouabain inhibition (I₀₅ = 8 μM) and corresponds to the enzyme conformation which is stimulated by the concurrent presence of Na⁺ and ATP or of Na⁺ and oligomycin. There appear to be two conformations of the enzyme that are regulated by Mg²⁺ binding on the inhibitory sites of the enzyme.     

Quantitative aspects of the interaction between ouabain and (Na + K)-activated ATPase in vitro

The inhibitory effect of ouabain on (Na⁺ + K⁺)-activated ATPase (Mg²⁺-dependent, (Na⁺ + K⁺)-activated ATP phosphohydrolase, EC 3.6.1.3) obtained from rat brain microsomal fraction was re-examined using a modified method to estimate the inhibited reaction velocity. This method involves a preincubation of a ouabain-enzyme mixture in the presence of Na⁺, Mg²⁺ and ATP to bring the ouabain-enzyme reaction to near equilibrium. The (Na⁺ + K⁺)-activated ATPase reaction was subsequently started by the addition of a KCl solution.     

Adenosine triphosphatase activity in the neural lobe of the bovine pituitary gland

1. Homogenates of neural lobes of bovine pituitary glands were fractionated by differential and density-gradient ultracentrifugation and the distribution of adenosine triphosphatase (ATPase) activity was studied. It was shown that all the activity was membrane-bound. 2. On the basis of ionic requirements the ATPase activity was grouped into three categories: (a) Mg²⁺-dependent, (b) Ca²⁺-dependent and (c) Mg²⁺+Na⁺+K⁺-dependent (ouabain-sensitive) ATPases. The activity in the absence of bivalent cations was negligible. The ratio between the activities of the three ATPases varied between the different subcellular fractions. 3. Preincubation of the subcellular fractions with deoxycholate increased the activity of the Mg²⁺+Na⁺+K⁺-dependent enzyme, whereas the Mg²⁺- and Ca²⁺-activated ATPases were either unaffected or slightly inhibited. Triton X-100 solubilized the Mg²⁺- and Ca²⁺-ATPases; however, the activity of the Mg²⁺+Na⁺+K⁺-ATPase was abolished by the concentration of Triton X-100 used. 4. All the subfractions displayed unspecific nucleotide triphosphatase activity towards GTP, ITP and UTP. These substrates inhibited the hydrolysis of ATP by all three ATPases. ADP also inhibited the ATPases. 5. Polyacrylamide-gel electrophoresis of extracts containing the Mg²⁺- and Ca²⁺-dependent ATPase activity solubilized by Triton X-100 revealed the presence of two enzymes; one activated by either Mg²⁺ or Ca²⁺ and the other activated only by Ca²⁺. 6. In sucrose density gradients the distribution of vasopressin was different from that of all three types of ATPases. It is therefore suggested that the neurosecretory granules do not possess ATPase activity.